PKHD1L1, a gene involved in the stereocilia coat, causes autosomal recessive nonsyndromic hearing loss.

Identification of genes associated with nonsyndromic hearing loss is a crucial endeavor given the substantial number of individuals who remain without a diagnosis after even the most advanced genetic testing. PKHD1L1 was established as necessary for the formation of the cochlear hair-cell stereociliary coat and causes hearing loss in mice and zebrafish when mutated. We sought to determine if biallelic variants in PKHD1L1 also cause hearing loss in humans. Exome sequencing was performed on DNA of four families segregating autosomal recessive nonsyndromic sensorineural hearing loss. Compound heterozygous p.[(Gly129Ser)];p.[(Gly1314Val)] and p.[(Gly605Arg)];p[(Leu2818TyrfsTer5)], homozygous missense p.(His2479Gln) and nonsense p.(Arg3381Ter) variants were identified in PKHD1L1 that were predicted to be damaging using in silico pathogenicity prediction methods. In vitro functional analysis of two missense variants was performed using purified recombinant PKHD1L1 protein fragments. We then evaluated protein thermodynamic stability with and without the missense variants found in one of the families and performed a minigene splicing assay for another variant. In silico molecular modeling using AlphaFold2 and protein sequence alignment analysis were carried out to further explore potential variant effects on structure. In vitro functional assessment indicated that both engineered PKHD1L1 p.(Gly129Ser) and p.(Gly1314Val) mutant constructs significantly reduced the folding and structural stabilities of the expressed protein fragments, providing further evidence to support pathogenicity of these variants. Minigene assay of the c.1813G>A p.(Gly605Arg) variant, located at the boundary of exon 17, revealed exon skipping leading to an in-frame deletion of 48 amino acids. In silico molecular modeling exposed key structural features that might suggest PKHD1L1 protein destabilization. Multiple lines of evidence collectively associate PKHD1L1 with nonsyndromic mild-moderate to severe sensorineural hearing loss. PKHD1L1 testing in individuals with mild-moderate hearing loss may identify further affected families.

Ex vivo expansion of hematopoietic stem cells in two/ three-dimensional co-cultures with various source of stromal cells.

The ex vivo expansion of hematopoietic stem cells, with both high quantities and quality, is considered a paramount issue in cell and gene therapy for hematological diseases. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells reveal the importance of using 2D and 3D coculture as a physiological system simulator in the proliferation, differentiation, and homeostasis of HSCs. Herein, the capacity of mesenchymal stem cells derived from different sources to support the expansion and maintenance of HSPC was compared with each other. We evaluated the fold increase of HSPC, CD34 marker expression, cytokine secretion profile of different MSCs, and the frequency of hematopoietic colony-forming unit parameters. Our results show that there was no significant difference between adipose tissue-MSC, Wharton jelly-MSC, and Endometrial-MSCs in HSPC expansion (fold increase: 34.74±4.38 in Wj-MSC, 32.22±5.07 in AD-MSC, 25.9±1.27 in En-MSCs); However, there were significantly more than the expansion media alone (4.4±0.69). The results obtained from the cytokine secretion analysis also confirm these results. Also, there were significant differences in the clonogenicity of Wj-MSC, En-MSCs, and expansion media (CFU-GEMM: 7±1.73, 2.3±1.15, and 2.3±1.52), which indicated that Wj-MSC could significantly maintain the primitive state. As a result, using Wj-mesenchymal stem cells on a 3D coculture system effectively increases the HSPC expansion and maintains the colonization potential of hematopoietic stem cells.

Identification of new variants in patients with mucopolysaccharidosis in consanguineous Iranian families.

Introduction: Mucopolysaccharidoses are a group of lysosomal storage disorders that include seven types that are classified based on the enzymes that are disrupted. Malfunction of these enzymes leads to the accumulation of glycosaminoglycans (GAGs) in various tissues. Due to genetic and clinical heterogeneity, diagnosing and distinguishing the different types is challenging. Genetic methods such as whole exome sequencing (WES) and Sanger sequencing are accurate methods for detecting pathogenic variants in patients. Methods: Thirty-two cases of mucopolysaccharidosis, predominantly from families with consanguineous marriages, were genetically examined. Out of these, fourteen cases underwent targeted sequencing, while the rest underwent WES. The results of WES were analyzed and the pathogenicity of the variants was examined using bioinformatics tools. In addition, a segregation analysis within families was carried out. Results: In most cases, a pathogenic or likely pathogenic variant was detected. Sixteen previously reported variants and six new variants were detected in the known IDS (c.458G>C, c.701del, c.920T>G), GNS (c.1430A>T), GALNS (c.1218_1221dup), and SGSH (c.149T>C) genes. Furthermore, we discovered a c.259G>C substitution in the NAGLU gene for the first time in three homozygous patients. This substitution was previously reported as heterozygous. Except for the variants related to the IDS gene, which were hemizygous, all the other variants were homozygous. Discussion: It appears that the high rate of consanguineous marriages in the families being studied has had a significant impact on the occurrence of this disease. Overall, these findings could expand the spectrum of pathogenic variants in mucopolysaccharidoses. Genetic methods, especially WES, are very accurate and can be used alone or in conjunction with other diagnostic methods for a more precise and rapid diagnosis of mucopolysaccharidoses. Additionally, they could be beneficial for family screening and disease prevention.

ZSCAN10 deficiency causes a neurodevelopmental disorder with characteristic oto-facial malformations.

Neurodevelopmental disorders are major indications for genetic referral and have been linked to more than 1,500 loci including genes encoding transcriptional regulators. The dysfunction of transcription factors often results in characteristic syndromic presentations, however, at least half of these patients lack a genetic diagnosis. The implementation of machine learning approaches has the potential to aid in the identification of new disease genes and delineate associated phenotypes. Next generation sequencing was performed in seven affected individuals with neurodevelopmental delay and dysmorphic features. Clinical characterization included reanalysis of available neuroimaging datasets and 2D portrait image analysis with GestaltMatcher. The functional consequences of ZSCAN10 loss were modelled in mouse embryonic stem cells (mESC), including a knock-out and a representative ZSCAN10 protein truncating variant. These models were characterized by gene expression and Western blot analyses, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR), and immunofluorescence staining. Zscan10 knockout mouse embryos were generated and phenotyped. We prioritized bi-allelic ZSCAN10 loss-of-function variants in seven affected individuals from five unrelated families as the underlying molecular cause. RNA-Seq analyses in Zscan10-/- mESCs indicated dysregulation of genes related to stem cell pluripotency. In addition, we established in mESCs the loss-of-function mechanism for a representative human ZSCAN10 protein truncating variant by showing alteration of its expression levels and subcellular localization, interfering with its binding to DNA enhancer targets. Deep phenotyping revealed global developmental delay, facial asymmetry, and malformations of the outer ear as consistent clinical features. Cerebral MRI showed dysplasia of the semicircular canals as an anatomical correlate of sensorineural hearing loss. Facial asymmetry was confirmed as a clinical feature by GestaltMatcher and was recapitulated in the Zscan10 mouse model along with inner and outer ear malformations. Our findings provide evidence of a novel syndromic neurodevelopmental disorder caused by bi-allelic loss-of-function variants in ZSCAN10.

Development of a potent recombinant scFv antibody against the SARS-CoV-2 by in-depth bioinformatics study: Paving the way for vaccine/diagnostics development.

BACKGROUND: The SARS-CoV-2 has led to a worldwide disaster. Thus, developing prophylactics/therapeutics is required to overcome this public health issue. Among these, producing the anti-SARS-CoV-2 single-chain variable fragment (scFv) antibodies has attracted a significant attention. Accordingly, this study aims to address this question: Is it possible to bioinformatics-based design of a potent anti-SARS-CoV-2 scFv as an alternative to current production approaches? METHOD: Using the complexed SARS-CoV-2 spike-antibodies, two sets analyses were performed: (1) B-cell epitopes (BCEs) prediction in the spike receptor-binding domain (RBD) region as a parameter for antibody screening; (2) the computational analysis of antibodies variable domains (VH/VL). Based on these primary screenings, and docking/binding affinity rating, one antibody was selected. The protein-protein interactions (PPIs) among the selected antibody-epitope complex were predicted and its epitope conservancy was also evaluated. Thereafter, some elements were added to the final scFv: (1) the PelB signal peptide; (2) a GSGGGGS linker to connect the VH-VL. Finally, this scFv was analyzed/optimized using various web servers. RESULTS: Among the antibody library, only one met the various criteria for being an efficient scFv candidate. Moreover, no interaction was predicted between its paratope and RBD hot-spot residues of SARS-CoV-2 variants-of-Concern (VOCs). CONCLUSIONS: Herein, a step-by-step bioinformatics platform has been introduced to bypass some barriers of traditional antibody production approaches. Based on existing literature, the current study is one of the pioneer works in the field of bioinformatics-based scFv production. This scFv may be a good candidate for diagnostics/therapeutics design against the SARS-CoV-2 as an emerging aggressive pathogen.

Clarin-2 gene supplementation durably preserves hearing in a model of progressive hearing loss.

Hearing loss is a major health concern affecting millions of people worldwide with currently limited treatment options. In clarin-2-deficient Clrn2-/- mice, used here as a model of progressive hearing loss, we report synaptic auditory abnormalities in addition to the previously demonstrated defects of hair bundle structure and mechanoelectrical transduction. We sought an in-depth evaluation of viral-mediated gene delivery as a therapy for these hearing-impaired mice. Supplementation with either the murine Clrn2 or human CLRN2 genes preserved normal hearing in treated Clrn2-/- mice. Conversely, mutated forms of CLRN2, identified in patients with post-lingual moderate to severe hearing loss, failed to prevent hearing loss. The ectopic expression of clarin-2 successfully prevented the loss of stereocilia, maintained normal mechanoelectrical transduction, preserved inner hair cell synaptic function, and ensured near-normal hearing thresholds over time. Maximal hearing preservation was observed when Clrn2 was delivered prior to the loss of transducing stereocilia. Our findings demonstrate that gene therapy is effective for the treatment of post-lingual hearing impairment and age-related deafness associated with CLRN2 patient mutations.

TRAPPC6B biallelic variants cause a neurodevelopmental disorder with TRAPP II and trafficking disruptions.

Highly conserved transport protein particle (TRAPP) complexes regulate subcellular trafficking pathways. Accurate protein trafficking has been increasingly recognized to be critically important for normal development, particularly in the nervous system. Variants in most TRAPP complex subunits have been found to lead to neurodevelopmental disorders with diverse but overlapping phenotypes. We expand on limited prior reports on TRAPPC6B with detailed clinical and neuroradiologic assessments, and studies on mechanisms of disease, and new types of variants. We describe 29 additional patients from 18 independent families with biallelic variants in TRAPPC6B. We identified seven homozygous nonsense (n = 12 patients) and eight canonical splice-site variants (n = 17 patients). In addition, we identified one patient with compound heterozygous splice-site/missense variants with a milder phenotype and one patient with homozygous missense variants. Patients displayed non-progressive microcephaly, global developmental delay/intellectual disability, epilepsy and absent expressive language. Movement disorders including stereotypies, spasticity and dystonia were also observed. Brain imaging revealed reductions in cortex, cerebellum and corpus callosum size with frequent white matter hyperintensity. Volumetric measurements indicated globally diminished volume rather than specific regional losses. We identified a reduced rate of trafficking into the Golgi apparatus and Golgi fragmentation in patient-derived fibroblasts that was rescued by wild-type TRAPPC6B. Molecular studies revealed a weakened interaction between mutant TRAPPC6B (c.454C>T, p.Q152*) and its TRAPP binding partner TRAPPC3. Patient-derived fibroblasts from the TRAPPC6B (c.454C>T, p.Q152*) variant displayed reduced levels of TRAPPC6B as well as other TRAPP II complex-specific members (TRAPPC9 and TRAPPC10). Interestingly, the levels of the TRAPPC6B homologue TRAPPC6A were found to be elevated. Moreover, co-immunoprecipitation experiments showed that TRAPPC6A co-precipitates equally with TRAPP II and TRAPP III, while TRAPPC6B co-precipitates significantly more with TRAPP II, suggesting enrichment of the protein in the TRAPP II complex. This implies that variants in TRAPPC6B may preferentially affect TRAPP II functions compared to TRAPP III functions. Finally, we assessed phenotypes in a Drosophila TRAPPC6B-deficiency model. Neuronal TRAPPC6B knockdown impaired locomotion and led to wing posture defects, supporting a role for TRAPPC6B in neuromotor function. Our findings confirm the association of damaging biallelic TRAPPC6B variants with microcephaly, intellectual disability, language impairments, and epilepsy. A subset of patients also exhibited dystonia and/or spasticity with impaired ambulation. These features overlap with disorders arising from pathogenic variants in other TRAPP subunits, particularly components of the TRAPP II complex. These findings suggest that TRAPPC6B is essential for brain development and function, and TRAPP II complex activity may be particularly relevant for mediating this function.

Biallelic variants in SLC4A10 encoding a sodium-dependent bicarbonate transporter lead to a neurodevelopmental disorder.

PURPOSE: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na+-dependent HCO3- import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness. METHODS: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles. RESULTS: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants. CONCLUSION: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice.

Genotypic variants of the tetrahydrobiopterin (BH4) biosynthesis genes in patients with hyperphenylalaninemia from different regions of Iran.

BACKGROUND: Hyperphenylalaninemia (HPA) is a metabolic disorder classified into phenylalanine-4-hydroxylase (PAH) and non-PAH deficiency. The latter is produced by mutations in genes involved in the tetrahydrobiopterin (BH4) biosynthesis pathway and DNAJC12 pathogenetic variants. The BH4 metabolism, including de novo biosynthesis involved genes (i.e., guanosine 5'-triphosphate cyclohydrolase I (GTPCH/GCH1), sepiapterin reductase (SR/SPR), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS)), and two genes that play roles in cofactor regeneration pathway (i.e., dihydropteridine reductase (DHPR/QDPR) and pterin-4α-carbinolamine dehydratase (PCD/PCBD1)). The subsequent systemic hyperphenylalaninemia and monoamine neurotransmitter deficiency lead to neurological consequences. The high rate of consanguineous marriages in Iran substantially increases the incidence of BH4 deficiency. METHODS: We utilized the Sanger sequencing technique in this study to investigate 14 Iranian patients with non-PAH deficiency. All affected subjects in this study had HPA and no mutation was detected in their PAH gene. RESULTS: We successfully identified six mutant alleles in BH4-deficiency-associated genes, including three novel mutations: one in QDPR, one in PTS, and one in the PCBD1 gene, thus giving a definite diagnosis to these patients. CONCLUSION: In this light, appropriate patient management may follow. The clinical effect of reported variants is essential for genetic counseling and prenatal diagnosis in the patients' families and significant for the improvement of precision medicine.

Biallelic BORCS8 variants cause an infantile-onset neurodegenerative disorder with altered lysosome dynamics.

BLOC-one-related complex (BORC) is a multiprotein complex composed of eight subunits named BORCS1-8. BORC associates with the cytosolic face of lysosomes, where it sequentially recruits the small GTPase ARL8 and kinesin-1 and -3 microtubule motors to promote anterograde transport of lysosomes toward the peripheral cytoplasm in non-neuronal cells and the distal axon in neurons. The physiological and pathological importance of BORC in humans, however, remains to be determined. Here, we report the identification of compound heterozygous variants [missense c.85T>C (p.Ser29Pro) and frameshift c.71-75dupTGGCC (p.Asn26Trpfs*51)] and homozygous variants [missense c.196A>C (p.Thr66Pro) and c.124T>C (p.Ser42Pro)] in BORCS8 in five children with a severe early-infantile neurodegenerative disorder from three unrelated families. The children exhibit global developmental delay, severe-to-profound intellectual disability, hypotonia, limb spasticity, muscle wasting, dysmorphic facies, optic atrophy, leuko-axonopathy with hypomyelination, and neurodegenerative features with prevalent supratentorial involvement. Cellular studies using a heterologous transfection system show that the BORCS8 missense variants p.Ser29Pro, p.Ser42Pro and p.Thr66Pro are expressed at normal levels but exhibit reduced assembly with other BORC subunits and reduced ability to drive lysosome distribution toward the cell periphery. The BORCS8 frameshift variant p.Asn26Trpfs*51, on the other hand, is expressed at lower levels and is completely incapable of assembling with other BORC subunits and promoting lysosome distribution toward the cell periphery. Therefore, all the BORCS8 variants are partial or total loss-of-function alleles and are thus likely pathogenic. Knockout of the orthologous borcs8 in zebrafish causes decreased brain and eye size, neuromuscular anomalies and impaired locomotion, recapitulating some of the key traits of the human disease. These findings thus identify BORCS8 as a novel genetic locus for an early-infantile neurodegenerative disorder and highlight the critical importance of BORC and lysosome dynamics for the development and function of the central nervous system.

PKHD1L1, A Gene Involved in the Stereocilia Coat, Causes Autosomal Recessive Nonsyndromic Hearing Loss.

Identification of genes associated with nonsyndromic hearing loss is a crucial endeavor given the substantial number of individuals who remain without a diagnosis after even the most advanced genetic testing. PKHD1L1 was established as necessary for the formation of the cochlear hair-cell stereociliary coat and causes hearing loss in mice and zebrafish when mutated. We sought to determine if biallelic variants in PKHD1L1 also cause hearing loss in humans. Exome sequencing was performed on DNA of four families segregating autosomal recessive nonsyndromic sensorineural hearing loss. Compound heterozygous p.[(Gly129Ser)];p.[(Gly1314Val)] and p.[(Gly605Arg)];p[(Leu2818TyrfsTer5)], homozygous missense p.(His2479Gln) and nonsense p.(Arg3381Ter) variants were identified in PKHD1L1 that were predicted to be damaging using in silico pathogenicity prediction methods. In vitro functional analysis of two missense variants was performed using purified recombinant PKHD1L1 protein fragments. We then evaluated protein thermodynamic stability with and without the missense variants found in one of the families and performed a minigene splicing assay for another variant. In silico molecular modelling using AlphaFold2 and protein sequence alignment analysis were carried out to further explore potential variant effects on structure. In vitro functional assessment indicated that both engineered PKHD1L1 p.(Gly129Ser) and p.(Gly1314Val) mutant constructs significantly reduced the folding and structural stabilities of the expressed protein fragments, providing further evidence to support pathogenicity of these variants. Minigene assay of the c.1813G>A p.(Gly605Arg) variant, located at the boundary of exon 17, revealed exon skipping leading to an in-frame deletion of 48 amino acids. In silico molecular modelling exposed key structural features that might suggest PKHD1L1 protein destabilization. Multiple lines of evidence collectively associate PKHD1L1 with nonsyndromic mild-moderate to severe sensorineural hearing loss. PKHD1L1 testing in individuals with mild-moderate hearing loss may identify further affected families.

Novel homozygous variants in PRORP expand the genotypic spectrum of combined oxidative phosphorylation deficiency 54.

Biallelic hypomorphic variants in PRORP have been recently described as causing the autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a phenotypic spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy. Here, we report three additional families with homozygous missense PRORP variants with pleiotropic phenotypes. Each missense variant altered a highly conserved residue within the metallonuclease domain. In vitro mitochondrial tRNA processing assays with recombinant TRMT10C, SDR5C1 and PRORP indicated two COXPD54-associated PRORP variants, c.1159A>G (p.Thr387Ala) and c.1241C>T (p.Ala414Val), decreased pre-tRNAIle cleavage, consistent with both variants impacting tRNA processing. No significant decrease in tRNA processing was observed with PRORP c.1093T>C (p.Tyr365His), which was identified in an individual with leukodystrophy. These data provide independent evidence that PRORP variants are associated with COXPD54 and that the assessment of 5' leader mitochondrial tRNA processing is a valuable assay for the functional analysis and clinical interpretation of novel PRORP variants.

Preparation, Purification and Performance Evaluation of Polyclonal Antibody Against SARS-CoV-2 Produced in Rat.

Purpose: Among all known human coronaviruses, some viruses (e.g., SARS-CoV-2) cause severe pneumonia or even death. With the regard to its spread and the importance of its rapid identification/treatment, and because pAbs are relatively cheap, able to bind to more sites on antigens and even neutralize them, this study was done for the production and purification of anti-SARS-CoV-2 polyclonal antibodies (pAb) in rats. Methods: Viral antigen purification was performed by PEG/NaCl precipitation. The efficiency of the sucrose cushion method was also investigated to produce a purer antigen. Immunization was done and antibody purification was performed by ammonium sulfate precipitation (33%), dialysis, and ion-exchange chromatography. Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to verify the antibody specificity. All data were analyzed by SPSS software. Results: The results showed that the amount of concentrated virus increased with the increase of PEG concentration. Moreover, the sucrose cushion method increased its purity. Besides, induction of immune response in rats for pAb production with high titers was reached via these antigens and ELISA/western blot results indicated a suitable antibody-antigen interaction. Additionally, it was shown that ion-exchange chromatography could be a suitable technique for IgG purification. Conclusion: Herein, we presented a simple and cheap method for the purification of whole viral particles with relatively high quality. The results verified that these antigens could elicit a good immune response in the rat. The obtained pAbs showed a good specificity toward SARS-CoV-2 antigens. Accordingly, this study proposes a promising method for viral vaccine development.

High incidence of AZF duplications in clan-structured Iranian populations detected through Y chromosome sequencing read depth analysis.

The ampliconic region of the human Y chromosome consists of large duplicated sequences that can undergo non-allelic homologous recombination (NAHR), resulting in structural rearrangements that may cause infertility, especially when they occur in the azoospermia factor b/c (AZFb/c) region. Although AZF duplications have long been neglected due to the technical limitations of STS-based studies that focused mainly on deletions, recent next generation sequencing (NGS) technologies provided evidence for their importance in fertility. In this study, a NGS read depth approach was used to detect AZFb/c rearrangements in 87 Iranians from different ethnic groups. The duplication frequency in Iran proved to be twice as high as in the "1000 Genomes" dataset. Interestingly, most duplications were found in patrilineal ethnic groups, possibly as a consequence of their lower male effective population size which can counteract negative selection. Moreover, we found a large 8.0 Mb duplication, resulting in a fourfold increase in the copy number of AZFc genes, which to our knowledge is the largest duplication ever reported in this region. Overall, our results suggest that it is important to consider not only AZF deletions but also duplications to investigate the causes of male infertility, especially in patrilineal clan-based populations.

The Effect of Platelet-Rich Plasma on the Osteoblastic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stromal Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are cell populations that have the potential to proliferate and differentiate. The process of stem cell differentiation from pluripotent cells to bone cells requires general changes in their pattern of gene expression, the most well-known of which are changes in miRNA-dependent settings. Platelet-enriched plasma (PRP) releases growth factors that are mitogenic to mesenchymal cells and can accelerate the process of osteogenic differentiation. The aim of this study was to investigate the effect of PRP on the expression changes of Let-7a, mir-27a, mir-31, mir-30c, mir-21, and mir-106a during osteogenic differentiation. METHODS: MSCs were isolated from adipose tissue after abdominoplasty and evaluated by flow cytometry. The ef-fect of PRP (10%) on the process of osteogenic differentiation was determined by measuring the expression of Let-7a, mir-27a, mir-31, mir-30c, mir-21, and mir-106a using the real-time polymerase chain reaction (PCR) technique. RESULTS: The increase in Let-7a expression was significant on the 14th day compared to the 3rd day. mir-27a expression rose significantly on the 3rd day. The expression of mir-30 exhibited a significant increase on the 14th day. mir-21 expression was significantly enhanced on the 3rd day and was downregulated on the 14th day. mir-106a expression showed a significant decreasing tendency between days 3 and 14 in a time-dependent pattern. CONCLUSIONS: These findings indicate that PRP probably accelerates the process of differentiation into bone. PRP, as a biological catalyst, showed a clear and distinct impact on the miRNAs regulating bone differentiation of human mesenchymal cells.

Treatment of Cutaneous Leishmaniasis with Intense Pulsed Light: Is it Effective?

Background: Cutaneous leishmaniasis (CL) is a common parasitic disorder that is endemic in many countries. There is no completely effective treatment for this condition, but pentavalent antimony compounds are regarded as the main treatment. Different laser types have been used for treatment of CL with variable success but to our best knowledge, there is no published paper regarding use of intense pulsed light (IPL) for treatment of CL. Materials and Methods: In this randomized, single-blind clinical trial study, we compared the efficacy of intralesional glucantime alone versus intralesional glucantime plus weekly IPL for treatment of 54 patients with confirmed cutaneous leishmaniasis for a maximum of eight weeks as a randomized, clinical trial. Results: Although it was not statistically significant, combination treatment was more effective than intralesional glucantime alone (P > 0.05). However, the velocity of healing was significantly higher in the IPL plus intralesional glucantime alone versus glucantime alone. No side effect was observed in both groups. Discussion: To better evaluate the efficacy of IPL, more studies with a higher number of patients and using various IPL filters are recommended.

Seminal prolactin is associated with HSP90 transcript content in ejaculated spermatozoa.

OBJECTIVE: Evidence indicates that an imbalance between the production of reactive oxygen species and defense ability of antioxidants has clinical significance in the pathophysiology of male infertility. To investigate the role of seminal prolactin (PRL) in the fertilizing capacity of men, the present study evaluated the associations of seminal PRL levels with semen parameters and heat shock protein 90 (HSP90) transcript abundance in ejaculated spermatozoa. METHODS: We assessed seminal PRL levels and the abundance of HSP90 transcripts in ejaculated spermatozoa from normozoospermic donors (n=18) and infertile men (n=18). The transcript content of HSP90 in ejaculated spermatozoa was analyzed using real-time polymerase chain reaction. RESULTS: Seminal PRL concentrations in infertile patients were significantly lower (p=0.004) than in fertile controls. Seminal PRL showed relatively good diagnostic power for discriminating infertile men (area under the curve=0.776; 95% confidence interval, 0.568 to 0.934; p=0.005). Significant positive correlations were seen between seminal PRL levels and sperm count (r=0.400, p=0.016) and progressive motility (r=0.422, p=0.010). Infertile patients showed a significantly higher abundance of sperm HSP90 than fertile controls (p=0.040). Sperm HSP90 transcript abundance was negatively correlated with sperm progressive motility (r=0.394, p=0.018). Men with higher seminal PRL levels exhibited a lower abundance of sperm HSP90 transcripts. CONCLUSION: Our finding demonstrated associations among semen quality, seminal PRL levels, and the abundance of HSP90 transcripts in ejaculated spermatozoa. Seminal PRL may contribute to male fertility by maintaining the seminal antioxidant capacity and may have the potential to act as a diagnostic and prognostic biomarker.

Brain monoamine vesicular transport disease caused by homozygous SLC18A2 variants: A study in 42 affected individuals.

PURPOSE: Brain monoamine vesicular transport disease is an infantile-onset movement disorder that mimics cerebral palsy. In 2013, the homozygous SLC18A2 variant, p.Pro387Leu, was first reported as a cause of this rare disorder, and dopamine agonists were efficient for treating affected individuals from a single large family. To date, only 6 variants have been reported. In this study, we evaluated genotype-phenotype correlations in individuals with biallelic SLC18A2 variants. METHODS: A total of 42 affected individuals with homozygous SLC18A2 variant alleles were identified. We evaluated genotype-phenotype correlations and the missense variants in the affected individuals based on the structural modeling of rat VMAT2 encoded by Slc18a2, with cytoplasm- and lumen-facing conformations. A Caenorhabditis elegans model was created for functional studies. RESULTS: A total of 19 homozygous SLC18A2 variants, including 3 recurrent variants, were identified using exome sequencing. The affected individuals typically showed global developmental delay, hypotonia, dystonia, oculogyric crisis, and autonomic nervous system involvement (temperature dysregulation/sweating, hypersalivation, and gastrointestinal dysmotility). Among the 58 affected individuals described to date, 16 (28%) died before the age of 13 years. Of the 17 patients with p.Pro237His, 9 died, whereas all 14 patients with p.Pro387Leu survived. Although a dopamine agonist mildly improved the disease symptoms in 18 of 21 patients (86%), some affected individuals with p.Ile43Phe and p.Pro387Leu showed milder phenotypes and presented prolonged survival even without treatment. The C. elegans model showed behavioral abnormalities. CONCLUSION: These data expand the phenotypic and genotypic spectra of SLC18A2-related disorders.

Putative founder effect of Arg338* AP4M1 (SPG50) variant causing severe intellectual disability, epilepsy and spastic paraplegia: Report of three families.

Bi-allelic variants affecting one of the four genes encoding the AP4 subunits are responsible for the "AP4 deficiency syndrome." Core features include hypotonia that progresses to hypertonia and spastic paraplegia, intellectual disability, postnatal microcephaly, epilepsy, and neuroimaging features. Namely, AP4M1 (SPG50) is involved in autosomal recessive spastic paraplegia 50 (MIM#612936). We report on three patients with core features from three unrelated consanguineous families originating from the Middle East. Exome sequencing identified the same homozygous nonsense variant: NM_004722.4(AP4M1):c.1012C>T p.Arg338* (rs146262009). So far, four patients from three other families carrying this homozygous variant have been reported worldwide. We describe their phenotype and compare it to the phenotype of patients with other variants in AP4M1. We construct a shared single-nucleotide polymorphism (SNP) haplotype around AP4M1 in four families and suggest a probable founder effect of Arg338* AP4M1 variant with a common ancestor most likely of Turkish origin.

Expression, purification, and study on the efficiency of a new potent recombinant scFv antibody against the SARS-CoV-2 spike RBD in E. coli BL21.

Many efforts have been made around the world to combat SARS-CoV-2. Among these are recombinant antibodies considered to be suitable as an alternative for some diagnostics/therapeutics. Based on their importance, this study aimed to investigate the expression, purification, and efficiency of a new potent recombinant scFv in the E. coli BL21 (DE3) system. The expression studies were performed after confirming the scFv cloning into the pET28a vector using specific PCRs. After comprehensive expression studies, a suitable strategy was adopted to extract and purify periplasmic proteins using Ni2+-NTA resin. Besides the purified scFv, the crude bacterial lysate was also used to develop a sandwich ELISA (S-ELISA) for the detection of SARS-CoV-2. The use of PCR, E. coli expression system, western blotting (WB), and S-ELISA confirmed the functionality of this potent scFv. Moreover, the crude bacterial lysate also showed good potential for detecting SARS-CoV-2. This could be decreasing the costs and ease its utilization for large-scale applications. The production of high-quality recombinant proteins is essential for humankind. Moreover, with attention to the more aggressive nature of SARS-CoV-2 than other coronaviruses, the development of an effective detection method is urgent. Based on our knowledge, this study is one of the limited investigations in two fields: (1) The production of anti-SARS-CoV-2 scFv using E. coli [as a cheap heterologous host] in relatively high amounts and with good stability, and (2) Designing a sensitive S-ELISA for its detection. It may also be utilized as potent therapeutics after further investigations.